The pulmonary microvascular endothelial barrier function is controlled by the PGE₂-EP₄ signaling axis

Background

Endothelial cells, like gate-keepers of the vascular bed, can actively protect against the inflammatory process. Prostaglandin E₂ (PGE₂) could be one of the mediators that can promote the barrier function of endothelial cells. PGE₂ exerts its cellular effects by binding to four different E-prostanoid receptors (EP_1–4) that belong to the family of G protein-coupled receptors. This project aimed at characterizing the barrier-protective properties of PGE₂ and especially EP₄ receptor on human pulmonary microvascular endothelial cells (HMVEC-L).

Methods

The endothelial barrier properties were analyzed by measurements of transendothelial electrical resistance (TEER) by Endohm and by ECIS (Electric Cell substrate Impedance Sensing) devices. Morphological studies were performed with immunofluorescence microscopy. Different protein expressions were detected by flow cytometry. Leukocyte diapedesis was studied by performing transendothelial migration assays of neutrophils and eosinophils.

Results

We found that the EP₄ receptor is expressed on HMVEC-L. PGE₂ and the selective EP₄ receptor agonist (ONO-AE1-329) prevented the barrier-disrupting effect of thrombin on the endothelial monolayer, as it was visualized by VE-cadherin staining. Selective blocking of EP₄ receptors (EP₄ antagonist ONO-AE3-208) inhibited the protective effect of PGE₂ on endothelial monolayers. PGE₂ and the EP₄ agonist enhanced the regeneration of electrically wounded endothelial monolayers. The specificity of EP₄ receptor involvement was proven by using the EP₄ receptor antagonist and selective agonists for EP₂ and EP₃ receptors. PGE₂ and the EP₄ receptor agonist attenuated the TNF-α-induced up-regulation of E-selectin. Surprisingly, this effect was not affected by an adenylyl cyclase inhibitor, but inhibition of PKC activity reversed the effect of the EP₄ receptor agonist and PGE₂ at reducing the E-selectin expression. In the cell interaction assays, thrombin or TNF-α increased the permeability of endothelial monolayers which also enhanced the transmigration of neutrophils and eosinophils, respectively. These effects were prevented by the selective activation of EP₄ receptors.

Conclusions

Our data support the hypothesis that endothelial cells, as gate-keepers of the vessel wall can actively participate in the inflammatory process. We have shown that PGE₂, via activating EP₄ receptors, enhances the barrier function of the endothelium by protecting the endothelial adherent junctional network and preventing leukocyte diapedesis. Therefore, EP₄ agonists might be promising new therapeutic tools in treating inflammatory diseases.